Research Paper
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Acta Biochim Biophys Sin
2005,37: 728-736 |
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doi:10.1111/j.1745-7270.2005.00106.x |
Cloning and Alternative
Splicing Analysis of Bombyx mori Transformer-2 Gene using Silkworm EST
Database
Bao-Long NIU1, Zhi-Qi MENG1*, Yue-Zhi TAO1, Shun-Lin LU2, Hong-Biao WENG1, Li-Hua HE1, and Wei-Feng SHEN1
1 Sericultural Research
Institute, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;
2 Department of
Sericulture and Apiculture, College of Animal Sciences, Zhejiang University,
Hangzhou 310029, China
Received: April 11,
2005
Accepted: August
30, 2005
*Corresponding
author: Tel, 86-571-86404031; E-mail, [email protected]
Abstract We have identified Bombyx
mori transformer-2 gene (Bmtra-2) cDNA by blasting the EST
database of B. mori. It was expressed in the whole life of the male and
female silkworm and was observed as a band of 1.3 kb by Northern blot analysis.
By comparing corresponding ESTs to the Bmtra-2 DNA sequence, it was
revealed that there were eight exons and seven introns, and all splice sites of
exons/introns conformed to the GT/AG rule. Bmtra-2 pre-mRNA can produce
multiple mRNAs encoding six distinct isoforms of BmTRA-2 protein using an
alternative splicing pathway during processing. Six types of Bmtra-2
cDNA clones were identified by reverse transcription-polymerase chain reaction.
All isoforms of BmTRA-2 protein contain two arginine/serine-rich domains and
one RNA recognition motif, showing striking organizational similarity to Drosophila
TRA-2 proteins.
Key words EST database; transformer-2 gene; Bmtra-2;
alternative splicing; gene clone; Bombyx mori
The transformer-2 gene (tra-2) that encoded a
pre-mRNA splicing protein for sex differentiation was first cloned in Drosophila
[1-3].
Genetic studies have shown that the tra-2 gene plays a key role in the
metazoan sexual differentiation regulatory cascade in Drosophila. The
female-specific transformer protein (TRA) functions in combination with TRA-2
proteins to direct female-specific doublesex gene (dsx) pre-mRNA splicing
[4-6].
The tra-2 gene has also been discovered in mammals, chickens and insects
as an mRNA splicing factor [7-9]. It encodes a pre-mRNA splicing protein that consists of two
arginine/serine-rich domains (RS domains) and one ribonucleoprotein (RNP) type
RNA binding domain, also identified as the RNA recognition motif (RRM) [10-12].
Here we cloned the Bombyx mori transformer-2 gene (Bmtra-2)
by blasting its expressed sequence tag (EST) database and using the DNA
sequencing approach [13,14], and found it could produce six alternatively
spliced mRNAs encoding six isoforms homologous to Drosophila TRA-2.
Materials and Methods
Silkworm strain
Silkworm strain p50 was donated by the Sericultural Research
Institute, Zhejiang Academy of Agricultural Sciences (Hangzhou, China). Their
sexes were distinguished by detecting the W chromosome-specific
retrotransposable element (W-Samurai; GenBank accession number AB012905) with
primers Samurai-1B and Samurai-2B [15].
B. mori EST database blasting with
RRM
The cDNA sequence of B. mori that encoded an amino acid
peptide containing the RRM was selected as a probe to blast the B. mori
EST database (http://www.ncbi.nlm.nih.gov/dbEST/) for homologous clones,
using the BLAST 2.1 program (http://www.ncbi.nlm.nih.gov/blast/). This
approach led to the identification of an EST clone, CK496349. The deduced amino
acid sequence of CK496349 has an RRM domain. This EST clone was used for
further analysis by selecting the corresponding ESTs from the blast output and
extending these ESTs to a new contig for further cycles of EST blasting. We
continued to recycle the contig and blast the ESTs until no new ESTs were
identified to the extended sequence [16]. In the relative identified ESTs,
there were many alternatively spliced isoforms. The primer pairs listed in Table
1 were designed according to these EST sequences.
Cloning of Bmtra-2 DNA
sequence
The genomic DNA was extracted from silkworm posterior silk glands
with the phenol-chloroform extraction method and was used as the template for
polymerase chain reaction (PCR) with the primer pairs listed in Table 1.
PCR products were cloned into a T-A cloning site of pMD-T vector (TaKaRa,
Dalian, China) and sequenced. These sequenced fragments were joined without any
intervening sequence. The Bmtra-2 DNA sequence was obtained.
The B. mori genomic database (http://www.ncbi.nlm.nih.gov/BLAST/Genome/Insects.html)
was blasted with the cloned Bmtra-2 gene. The genomic clones that were
aligned with the Bmtra-2 DNA sequence were identified and joined. The
sequence upstream of the Bmtra-2 gene was obtained and put into the
promoter website (http://thr.cit.nih.gov/molbio/proscan/) to search the
promoter regions and putative transcription factor-binding (TFB) sites to
investigate its transcriptional regulation.
Analysis of alternative
splicing of Bmtra-2 pre-mRNA by reverse transcription (RT)-PCR
Total RNA was separately extracted from five kinds of tissues of 3-d
fifth instar male and female larvae using EASYPrep RNA (TaKaRa): the fat body,
Malpighian tubule, silk gland, testis or ovary. Poly(A)+ RNA
was isolated from the fat body using a Micro-FastTrack 2.0 mRNA isolation kit
(Invitrogen Corp., California, USA). The first-strand cDNA synthesis was
performed with an oligo(dT) primer (5'-TTTTTTTTTTTTTTTTTTTXX-3').
PCR reactions were done for testing the alternatively spliced mRNAs with the
primer pairs listed in Table 1 and the LA RNA PCR kit (TaKaRa). PCR
products were purified on a 1.5% agarose gel, and cloned into a T-A cloning
site of pMD-T vector and sequenced. All procedures were carried out according
to the protocol provided by the manufacturer.
Northern blot hybridization
Total RNA was isolated separately from the male and female silkworms
at different stages by EASYPrep RNA: egg, larval, pupal and adult. For Northern
blot hybridization, total RNAs were subjected to electrophoresis on a 1.2%
agarose gel in the presence of 2.2 mM formaldehyde and transblotted onto a
nylon membrane. The membrane was prehybridized and hybridized with the Bmtra-2
cDNA probe labeled with digoxigenin (Roche Corp., Mannheim, Germany) and
detected with Ap-anti digoxigenin according to the manufacturer�s instructions.
Results
In silico cloning of Bmtra-2
cDNA by blasting silkworm EST database
It has been reported previously that TRA-2 is an RNA binding protein
containing an RRM. In the B. mori EST database, there was a clone,
CK496349, with strong similarity to RRM in its deduced amino acid sequence. It
was used as the probe for further analysis by electing the corresponding ESTs
from the blast output and extending to a new contig for further cycles of EST
blasting. A total of 27 ESTs were obtained, as shown in Table 2. These
ESTs can be assembled to many contigs with a complete open reading frame
encoding proteins that contain two RS domains at each end and the same RRM
found in CK496349. The seven-glycine (G) region was located between the RRM and
the C-terminal RS domain. One isoform was PA, the nucleotide and deduced amino
acid sequences of which are shown in Fig. 1. The overall organization of
these proteins was similar to the Drosophila TRA-2264
and to many other RNA binding proteins. PA showed 80% homology to that of Apis
mellifera (GenBank accession number XP_396858), 68% to that of Bactrocera
oleae (CAD67988), 65% to that of Drosophila virilis
(AAB58113), 64% to that of Musca domestica (AAW34233), 60% to
that of Drosophila melanogaster (AAA28953) and 58% to that of Anopheles
gambia (EAA13826) (Fig. 2).
The conserved regions were the RRM and RRM-linker junction region.
Although similarity extended throughout the entire protein, it should be noted
that the RS domains were of low sequence complexity, diminishing the
significance of the matches in these regions. The RS domain sequences in the
silkworm and the fly TRA-2 proteins aligned only in areas of alternating
arginines and serines, suggesting that the arginine/serine-rich composition of
these domains, rather than the primary sequence, is conserved. In addition,
there was a glycine-rich region similar to that of human TRA-2a (hTRA-2a; GenBank
accession number AAC50658) [8], but it was not contained in the known TRA-2
proteins of other insects (Fig. 2). Based on the organizational and
sequence similarities of this silkworm TRA-2 to Drosophila TRA-2, we
designated this gene the silkworm Bmtra-2.
Bmtra-2 gene structure
To determine the exon/intron organization of the Bmtra-2
gene, its DNA sequence was obtained (GenBank accession number AY626066) by
combining many PCR fragments with no intervening sequences. The PCR fragments
were produced with the silkworm genomic DNA as the template and the primer
pairs were designed according to the sequence of Bmtra-2 cDNA (Table
1). It was revealed that there were eight exons and seven introns in the Bmtra-2
gene. All splicing sites of exons/introns conformed to the GT/AG rule.
Three genomic clones, BAAB01121891, BAAB01077090 and BAAB01073639,
were obtained by blasting the B. mori genomic database with the Bmtra-2
DNA sequence. They overlapped with each other and all showed a perfect match
with the Bmtra-2 DNA sequence in the corresponding regions. The sequence
upstream of the Bmtra-2 gene was included in the clone BAAB01121891. The
promoter region was on the forward strand between -2007 and -1757 bp upstream
of the Bmtra-2 gene transcriptional initiation site. A TATA-like element
was at -1785 bp. The positions of other putative TFB sites are also shown in
Table 3. Whether the presence of these sites is relevant to the
transcriptional regulation of the Bmtra-2 gene remains to be analyzed in
future studies.
Alternative splicing of Bmtra-2
pre-mRNA
By comparing the sequences of the resulting 27 ESTs from blasting
the B. mori EST database with the RRM to the Bmtra-2 DNA
sequence, three acceptor sites were found in the second intron and two acceptor
sites in the seventh intron. The nucleotide sequences of alternative splicing
ESTs from Bmtra-2 pre-mRNA are shown in Fig. 3(A,B). The specific
primers were designed according to the two alternative splicing sites, and six
specific primer pairs were used for detecting the different transcripts. All
six RT-PCR reactions with mRNAs, which were extracted separately from five
organs of 3-d fifth instar male and female larvae (the fat body, Malpighian
tubule, silk gland, testis and ovary) showed positive results [Fig. 3(C)].
These results indicated that there are six mRNAs produced from Bmtra-2
pre-mRNA using the alternative splicing pathway. The gene structure of Bmtra-2
and its six alternatively spliced mRNAs are shown in Fig. 4, with
deduced amino acid numbers in parentheses.
All six isoforms (PA, PB, PC, PD, PE and PF) deduced from six
alternative splicing mRNAs of Bmtra-2 pre-mRNA contained two RS domains
at each end and one RRM. There were no major differences between them. PD, PE
and PF have a different C-terminus, with a tyrosine phosphorylation site,
compared with PA, PB and PC. PA and PD have 11 amino acid residues more than PB
and PE respectively; and have 15 amino acid residues more than PC and PF,
respectively. PA, PB, PD and PE have one threonine phosphorylation site more
than PC and PF respectively. Whether these differences in the phosphorylation
site bring about different roles will be analyzed in the future.
Northern blot hybridization
To determine the size of Bmtra-2 mRNA transcripts, Northern
blot analysis was conducted using the digoxigenin-labeled product produced by
PCR reaction, with digoxigenin as the probe. Only a band of nearly 1.3 kb was
observed. It was expressed in all stages of eggs, larvae, pupas and adults of
the male and female silkworm (Fig. 5). According to the results of the in
silico clone and RT-PCR reaction, there should be six isoforms of Bmtra-2
mRNA. However, these isoforms had no significant differences in length and
could not be distinguished using polyacrylamide gel electrophoresis, so that
the six RT-PCR reactions with six different pairs of primers seemed to possess
the same band. It is for this reason that there was only one band in Northern
blot hybridization.
Discussion
In this report, we used a bioinformatic (or in silico)
strategy to quickly clone and identify the Bmtra-2 gene. This is
different from the time-consuming, traditional homologous gene cloning
approach, which needs degenerate-priming RT-PCR or low stringency screening of
both the cDNA and genomic libraries of silkworm. The bioinformatic approach
takes advantage of genetic and sequence information available for silkworm from
public databases [13,14]. By searching those databases with the blast program,
it was found that the silkworm EST clone CK496349 contains an RRM that is
similar to the corresponding domain of RNA-binding proteins. Through further
blasting, many cDNA sequences identified to the original sequence were
obtained. The genomic DNA sequence was also cloned. By comparing all the
relative ESTs to the DNA sequence, it was revealed that there were eight exons
and seven introns in the Bmtra-2 gene, and three acceptor sites in the
second intron and two acceptor sites in the seventh intron. RT-PCR reactions
with six different pairs of the specific primers, designed according to the two
alternative splicing sites, revealed that there are six mRNAs produced from
Bmtra-2 pre-mRNA using the alternative splicing pathway in all tested
tissues of the male and female silkworm. Six isoforms all contain one RRM similar
to the corresponding domain of RNA binding proteins and two RS domains
at each end. These organizations were similar to that of TRA-2 [10-12]. The most
conserved regions were the RRM and RRM-linker junction regions. The
similarities in the RS domains are low, as the matches in these regions align
only in areas of alternating arginines and serines. The phosphorylation sites
are different among the six isoforms. They may have different rules to affect
the splicing of different
pre-mRNAs in silkworm.
In Drosophila, tra-2 pre-mRNA can produce multiple
mRNAs encoding three distinct isoforms of TRA-2 protein (TRA-2264,
TRA-2226 and TRA-2179) using the alternative splicing
pathway during development [2,5]. The tra-2 gene plays a key role in the
�sex-determination
cascade. TRA-2 is one of the two factors known from genetic analysis to be
directly required for processing of dsx pre-mRNA along the
female-specific pathway in Drosophila [4-6]. It functions in
combination with TRA to direct female-specific dsx splicing [17,18].
B. mori dsx (Bmdsx) acts as a
double-switch gene at the final step in the sex-determination cascade in the
same way as in Drosophila dsx [19]. Although Bmtra-2
can produce multiple mRNAs encoding six distinct isoforms just like that of tra-2
in Drosophila, BmTRA-2 proteins do not seem to be required in the
sex-specific splicing of Bmdsx pre-mRNA, because the TRA/TRA-2 binding
motif-related sequence is not present in the Bmdsx genomic sequence, and
Bmdsx pre-mRNA processing would need splicing repressor(s) rather than
splicing activator(s), such as TRA and TRA-2 [20-23]. Given that Bmtra-2
can not affect Bmdsx pre-mRNA splicing, it is surprising that the RRM,
which is thought to constitute the major RNA binding domain for this protein,
is only 65% identical to the Drosophila virilis TRA-2 (Fig. 2).
Of the 71 identical residues in the RRM, 32 are conserved at the positions
that make up the RRM consensus (Fig. 1) and thus are very similar to
sequences found in many proteins that do not interact specifically with dsx
pre-mRNA, such as the RRM in the U1A and U2B'' proteins which contain
residues shown to be essential for RNA binding specificity [24-26]. The
RRM-linker junction region is similar to the known TRA-2 proteins in other
insects and is likely to perform conserved functions that are specific to
TRA-2. But the seven-glycine region similar to that of hTRA-2a is not
contained in other insects' known TRA-2 protein (Fig. 2). hTRA-2a protein is able
to recognize and affect the splicing of the dsx pre-mRNA in a manner to
that of TRA-2 expressed in Drosophila [8]. However, there have been no
natural human targets for hTRA-2a found in the human genome. HTRA-2b (GenBank
accession number AAB08701), another human SR-like splicing factor and human
homolog of Drosophila tra-2, which has many isoforms generated by
alternative splicing [27,28], is involved in the regulation of alternative
splicing processes during neural development, particularly the splicing of
fibroblast growth factor receptor 2 (FGF-2R) and glutamate receptor subunit B
(GluR-B) genes. The results therefore suggest that TRA-2b plays an
important role in neural differentiation by regulating the FGF-2R and GluR-B
genes [29,30]. So it can be proposed that BmTRA-2 may interact with specific
silkworm pre-mRNAs to affect their splicing patterns, just as hTRA-2 does, in
a manner analogous to the way TRA-2 affects dsx splicing.
Acknowledgements
This work was conducted in the Laboratory of Entomo-Molecular
Biology, Zhejiang Academy of Agricultural Sciences (Hangzhou, China).
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